Method and apparatus for detecting chemical binding

ABSTRACT

The method for screening binding between a target binder and potential pharmaceutical chemicals involves sending a solution (preferably an aqueous solution) of the target binder through a conduit to a size exclusion filter, the target binder being too large to pass through the size exclusion filter, and then sending a solution of one or more potential pharmaceutical chemicals (preferably an aqueous solution) through the same conduit to the size exclusion filter after target binder has collected on the filter. The potential pharmaceutical chemicals are small enough to pass through the filter. Afterwards, x-rays are sent from an x-ray source to the size exclusion filter, and if the potential pharmaceutical chemicals form a complex with the target binder, the complex produces an x-ray fluorescence signal having an intensity that indicates that a complex has formed.

STATEMENT REGARDING FEDERAL RIGHTS

This invention was made with government support under Contract No.W-7405-ENG-36 awarded by the U.S. Department of Energy. The governmenthas certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates generally to a flow method for detectingchemical binding and characterizing the binding between a chemical and atarget binder using x-ray fluorescence spectroscopy.

BACKGROUND OF THE INVENTION

The binding properties of a protein largely depend on the exposedsurface amino acid residues of its polypeptide chain (see, for example,Bruce Alberts et al., “Molecular Biology of the Cell”, 2^(nd) edition,Garland Publishing, Inc., New York, 1989; and H. Lodish et al.,“Molecular Cell Biology”, 4^(th) edition, W. H. Freeman and Company,2000). These amino acid residues can form weak noncovalent bonds withions and molecules. Effective binding generally requires the formationof many weak bonds at a “binding site,” which is usually a cavity in theprotein formed by a specific arrangement of amino acids. There must be aprecise fit with the binding site for effective binding to occur.

Pharmaceutical chemicals are the active ingredients in drugs, and it isbelieved that their therapeutic properties are linked to their abilityto bind to one or more binding sites. The shapes of these binding sitesmay differ greatly among different proteins, and even among differentconformations of the same protein. Even slightly different conformationsof the same protein may differ greatly in their binding abilities. Forthese reasons, it is extremely difficult to predict which chemicals willbind effectively to proteins. Research and development for a newpharmaceutical chemical for a drug, i.e. drug development, generallyinvolves determining the binding affinities between a potentialpharmaceutical chemical (preferably a water soluble organic chemicalthat can dissolve into the blood stream) and a target binder (generallya biological material such as an enzyme or non-enzyme protein, DNA, RNA,human cell, plant cell, animal cell, and the like) at many stages of thedrug development process. The target binder may also be a microorganism(e.g. prion, virus, bacterium, spores, and the like) in whole or inpart. The drug development process typically involves procedures forcombining potential pharmaceutical chemicals with target binders,detecting chemical binding between the potential pharmaceuticalchemicals and the target binders and determining the binding affinityand kinetics of binding of a target binder to a chemical to form acomplex or the kinetics of release of a bound chemical from a complex.The binding affinity is defined herein as the associative equilibriumconstant Ka, where Ka is defined by equation (1) below.Ka=[complex]/[target binder][potential pharmaceutical chemical]  (1)In equation (1), [complex] is the concentration in moles per liter ofthe target binder/potential pharmaceutical complex, [target binder] isthe concentration in moles per liter of the target binder, and[potential pharmaceutical chemical] is the concentration in moles perliter of the potential pharmaceutical chemical. Nowadays, the drugdevelopment process may involve the rapid screening of hundreds orthousands of potential pharmaceutical chemicals in order to identify a“lead compound,” which is one of the many tested that binds verystrongly, i.e. has a high binding affinity, with a particular targetbinder. After such a lead compound has been identified, then otherpotential pharmaceutical chemicals similar in structure to the leadcompound are synthesized and tested in order to determine which of thesepotential pharmaceutical chemicals, if any, exhibits an even higherbinding affinity. Some screening methods are described in the followingpatents, all of which are hereby incorporated by reference.

U.S. Pat. No. 6,147,344 to D. Allen Annis et al. entitled “Method forIdentifying Compounds in a Chemical Mixture”, which issued Nov. 14,2000, describes a method for automatically analyzing mass spectrographicdata from mixtures of chemical compounds.

U.S. Pat. No. 6,344,334 to Jonathan A. Ellman et al. entitled“Pharmacophore Recombination for the Identification of Small MoleculeDrug Lead Compounds,” which issued Feb. 5, 2002, describes a method foridentifying a drug lead compound by contacting target biologicalmolecules with cross-linked binding fragments.

U.S. Pat. No. 6,395,169 to Ole Hindsgaul et al. entitled “Apparatus forScreening Compound Libraries,” which issued May 28, 2002, describes anapparatus that employs frontal chromatography combined with massspectrometry to identify and rank members of a library that bind to atarget receptor.

Some screening methods (fluorescence activated cell sorting, forexample) cannot detect binding between many types of potentialpharmaceutical chemicals (a non fluorescent antibody, for example) and atarget binder unless the potential pharmaceutical chemical is providedwith a chemical tag. Tagging may involve modifying the original chemicalby attaching a “tag” (a chemical group that fluoresces when exposed toultraviolet or visible light, for example) to a portion of the potentialpharmaceutical chemical. Afterwards, the tagged chemicals are exposed tocells (muscle cells, for example) for a long enough period of time forbinding to occur (if it does occur) to some of the cells to produce acell/antibody complex. Afterwards, the cells are sent through a laserbeam one at a time. The laser producing the beam is interfaced to adetector, such as an ultraviolet/visible fluorescence detector. Thecell/antibody complexes produce a detectable fluorescence signal whenexposed to the laser beam, and as the screening method proceeds, when afluorescence signal is detected, the bound complexes that produce thesignal are collected (see, for example, Bruce Alberts et al., “MolecularBiology of the Cell”, 2^(nd) edition, Garland Publishing, Inc., NewYork, 1989, pages 159-160).

Another form of tagging involves attachment of the target binder to asurface. This type of tagging is used with surface plasmon resonancetechniques (see, for example, U.S. Pat. No. 5,641,640 to A. Hanningentitled “Method of Assaying for an Analyte Using Surface PlasmonResonance,” which issued Jun. 24, 1997; and U.S. Pat. No. 5,595,456 toM. Malmqvist et al entitled “Analyte Detection,” which issued Oct. 12,1999, both hereby incorporated by reference).

It is generally assumed that the attachment of a fluorescent tag onlyserves to make visible to the instrument the otherwise invisiblechemical and/or target binder, and that binding properties of the taggedand untagged materials are exactly the same. These assumptions may notbe valid, as it is well known that even small changes to the structureof a chemical or target binder may affect its function. Tagged materialsare structurally different from their untagged counterparts, and thesestructural differences could affect their binding properties.

An efficient method for screening chemicals (with potentialpharmaceutical activity, for example) for binding to target bindersremains highly desirable.

Therefore, an object of the present invention is to provide an efficientmethod for screening chemicals for binding to target binders.

Another object of the present invention is to provide a screening methodthat does not require modification of a potential pharmaceuticalchemical with a chemical tag.

Additional objects, advantages and novel features of the invention willbe set forth in part in the description which follows, and in part willbecome apparent to those skilled in the art upon examination of thefollowing or may be learned by practice of the invention. The objectsand advantages of the invention may be realized and attained by means ofthe instrumentalities and combinations particularly pointed out in theappended claims.

SUMMARY OF THE INVENTION

In accordance with the objects and purposes of the present invention, asembodied and broadly described herein, the present invention includes ascreening method that involves sending a fluid that includes targetbinder through a conduit to a size exclusion filter, the target binderbeing too large to pass through the size exclusion filter; sending asecond fluid that includes one or more chemicals through the conduit,the one or more chemicals capable of potentially binding to the targetbinder, the one or more chemicals being small enough to pass through thefilter; sending x-rays into the conduit near the size exclusion filter;and monitoring any x-ray fluorescence signal produced from inside theconduit near the size exclusion filter.

The invention also includes an apparatus for screening binding between atarget binder and a chemical. The apparatus includes an x-raytranslucent conduit for fluid having target binder and chemical. Afilter inside the conduit substantially blocks the flow of the targetbinder through the conduit but not the flow of the chemical. Theapparatus also includes an x-ray excitation source capable of sending anx-ray beam through a volume of fluid inside the conduit, and an x-raydetector capable of receiving an x-ray fluorescence signal produced fromthe volume of fluid inside said conduit exposed to the x-ray beam.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and form a part ofthe specification, illustrate the embodiment(s) of the present inventionand, together with the description, serve to explain the principles ofthe invention. In the drawings:

FIG. 1 shows a schematic representation of the apparatus of theinvention;

FIG. 2 shows a schematic representation of the apparatus of FIG. 1 astarget binder is admitted and collects on the size exclusion filter; and

FIG. 3 shows a schematic representation of the apparatus of FIG. 2 aftera potential pharmaceutical chemical binds to target binder.

DETAILED DESCRIPTION OF THE INVENTION

The present invention includes a method for detecting and measuringbinding affinities between chemicals and target binders. The methodinvolves sending a solution (preferably an aqueous solution) of targetbinder through a conduit to a size exclusion filter. The target bindersused with the invention are too large to pass through the size exclusionfilter. After sending the target binder into the conduit, the amount oftarget binder near the size exclusion filter, upstream of the sizeexclusion filter, and downstream of the size exclusion filter may bequantified by sending x-rays to these sections and detecting any x-rayfluorescence signal due to the target binder. If an x-ray fluorescencesignal due to the target binder is detected downstream of the particularfilter used, then the filter may be damaged or the pore size may not besmall enough to block the flow of target binder. After target bindercollects on the size exclusion filter, a solution (preferably an aqueoussolution) of one or more potential pharmaceutical chemicals is sentthrough the same conduit. The potential pharmaceutical chemicals usedwith the invention should be small enough to pass through the filter.The amount of the chemical(s) near the size exclusion filter, upstreamof the size exclusion filter, and downstream of the size exclusionfilter may be similarly quantified by x-ray fluorescence. Bindingbetween target binder and a chemical to form a complex can occuranywhere along the conduit where target binder and chemical are present,most likely near the size exclusion filter where target binder collects.

The invention also includes an apparatus for detecting and measuringbinding affinities between potential pharmaceutical chemicals and targetbinders. The apparatus includes a conduit and a size exclusion filterinside the conduit that is selected for passing the potentialpharmaceutical chemical(s) but not the target binder(s). The apparatusalso includes an x-ray source and an x-ray fluorescence detector.

The apparatus may include a collector for collecting materials that exitthe conduit. Reservoirs may also be provided for the solutions of targetbinder, potential pharmaceutical chemicals, and for solvents and othertypes of solutions such as buffer solutions and solutions of denaturingand/or decomplexing agents.

Solutions of decomplexing agents can be used with the invention. Asolution of decomplexing agent, for example, can be released into theconduit to promote the separation of a complex into its components oftarget binder and unbound chemical. After the chemical is released,another solution of potential pharmaceutical chemical (the chemical at adifferent concentration, a different chemical, etc.) can be introducedinto the conduit and screened for binding to the same target binder.

Preferably, the apparatus is under the control of a programmablecomputer using software that can be used to control the apparatus bycontrolling the flow of target binder solution into the conduit,controlling the flow of solution of one or more selected potentialpharmaceutical chemicals into the conduit, controlling the x-ray source(adjusting the position of the beam, the strength of the beam, etc.)receiving information related to any fluorescence signals received bythe x-ray detector. The flow of any other solvents or solutions can alsobe under computer control.

The invention uses x-ray fluorescence to measure the binding affinity ofa complex. The x-ray fluorescence is an important aspect of theinvention because each chemical element has its own unique x-rayfluorescence spectrum and the intensity of the fluorescence is relatedto the concentration of that element in a chemical sample. Briefly, whenan atom of a particular element is irradiated with x-ray radiation, theatom ejects a core electron such as a K shell electron. The resultingatom is in an excited state, and it can return to the ground state byreplacing the ejected electron with an electron from a higher energyorbital. This is accompanied by the emission of an x-ray photon, i.e.x-ray fluorescence, and the photon energy is equal to the difference inthe energies of the two electrons. Each element has a characteristic setof orbital energies and therefore, a characteristic x-ray fluorescencespectrum.

The use of x-ray fluorescence to detect binding events between targetbinders and receptors has been described in U.S. patent application No.20030027129 to Benjamin P. Warner et al., entitled “Method for DetectingBinding Events Using Micro-X-ray Fluorescence Spectrometry,” which waspublished Feb. 6, 2003, and in U.S. patent application Ser. No.10/206,524 to George J. Havrilla et al. entitled “Flow Method andApparatus for Screening Chemicals Using Micro-X-Ray Fluorescence,” bothhereby incorporated by reference. The use of capillary electrophoresiswith x-ray fluorescence has been described by T. C. Miller et al. in“Capillary Electrophoresis Micro X-ray Fluorescence: A Tool for BenchtopElemental Analysis,” Analytical Chemistry, vol. 75, pp. 2048-2053,(2003); and by M. C. Ringo et al. in “On-line X-ray FluorescenceDetection for Capillary Electrophoresis Separations,” NuclearInstruments and Methods in Physics Research B, vol. 149, pp. 177-181,1999; and by S. E. Mann et al. in “Element-Specific Detection inCapillary Electrophoresis Using X-Ray Fluorescence Spectroscopy,”Analytical Chemistry, vol. 72, pp. 1754-1758, (2000), incorporated byreference herein. Mann et al. report the preparation of a mixture ofchelation complexes of CDTA (cyclohexane diamine tetraacetic acid) andsubsequent separation using capillary electrophoresis. The separatedcomplexes were detected using a synchrotron-generated monochromatic, 10keV x-ray beam.

Many popular drugs include pharmaceutical chemicals that contain theelements fluorine, chlorine, and/or sulfur. X-ray fluorescencespectrometry is especially suited for detecting these types of potentialpharmaceutical chemicals because it can be used to detect and quantifythese elements, and in general, it can provide information about thequantity of an element with an atomic number of nine or higher. Inprinciple, x-ray fluorescence spectrometry can be used to provide thequantity of any element.

The practice of the invention can be further understood with theaccompanying figures. Similar or identical structure is identified usingidentical callouts. FIG. 1 shows a schematic representation of anembodiment of the apparatus of the invention. Apparatus 10 includesconduit 12, preferably a capillary tube, which is translucent to atleast some types of x-rays. Conduit 12 can be made from polyethylene,polypropylene, polytetrafluoroethylene (TEFLON™), polycarbonate, boronnitride, and other materials that are composed of elements having lowatomic numbers.

The material and thickness used for conduit 12 can be selected foroptimizing the x-ray fluorescence signal and durability of the conduit.The conduit wall should be as thin as practical for maximizing thetranslucency. Material translucencies for a thickness that attenuatescharacteristic K_(α) x-rays of phosphorus, sulfur, chlorine, fluorine,nitrogen, and carbon by a factor of 1/e are listed in Table 1.Preferably, the conduit walls can attenuate the x-ray fluorescencesignal by less than a factor of about 10,000. These elements were chosenbecause they are often present in known pharmaceutical chemicals. TABLE1 Thickness in Microns of Material That Attenuates Elemental X-rays by1/e Poly- Boron Material Polyethylene Teflon carbonate nitride BerylliumPhosphorus 52 7 28 16 88 Sulfur 80 11 42 24 136 Chlorine 120 16 63 37210 Fluorine 2.1 0.4 1.2 0.7 3 Nitrogen 0.6 0.6 0.5 0.2 0.7 Carbon 0.250.24 0.2 0.27 0.27

Size exclusion filter 14 remains stationary inside conduit 12. Sizeexclusion filter 14 includes pores that are small enough to block theflow of the target binders used with the invention but large enough notto block the flow of potential pharmaceutical chemicals being tested forbinding to the target binder. For detecting binding between a proteinand a potential pharmaceutical chemical, size exclusion filter 14 ispreferably a cellulose-based filter (an ULTRACEL™ AMICON™ YM10Ultrafiltration Disc, for example) or a mesoporous inorganic materialsuch as a zeolite (MCM-41 for example). The key attribute of sizeexclusion filter 14 is that it blocks the flow of the target binderwhile not blocking the flow of the potential pharmaceutical chemicalbeing tested. Size exclusion filter 14 can have any shape (e.g. flat,curved, round, etc.) or orientation (e.g. perpendicular as shown inFIGS. 1-3 or at other angles) relative to conduit 12.

Apparatus 10 also includes an x-ray excitation source 16 and an x-raydetector 18. X-ray excitation source 16 sends a beam of x-rays(preferably a collimated and focused beam) to a section of conduit 12.FIG. 1 shows x-ray source pointing at a section near size exclusionfilter 14. In practice, x-ray excitation source 16 can be used to obtaina baseline measurement of the concentration of potential pharmaceuticalchemical along conduit 12 by sending x-rays to various sections ofconduit 12 and detecting the x-ray fluorescence using x-ray detector 18.The wavelength and/or energy of the x-ray fluorescence is used toidentify a particular element present in the target binder and/orpotential pharmaceutical, and the intensity of the signal is used to forquantification. The x-ray excitation source 16 and x-ray detector 18used to demonstrate the invention were part of a commercially availablespectrometer, the EDAX Eagle II XPL energy dispersive x-ray fluorescencespectrometer, equipped with a microfocus x-ray tube, lithium driftedsilicon solid-state detector, processing electronics, and vendorsupplied operating software. The x-ray tube provided the source ofx-rays, and the lithium drifted silicon detector provided the x-rayfluorescence detector.

While an x-ray tube is the preferred x-ray source of the invention forreasons of cost and ease of use, it should be understood that anyexcitation source that produces an x-ray fluorescence signal from thetarget binder, potential pharmaceutical, and complex may be used. Inprinciple, a proton accelerator that is typically used forproton-induced x-ray emission (PIXE) and proton-induced gamma-rayemission (PIGE) may be used with the present invention. PIXE excitationis described by C. Vogt et al. in “Element Sensitive X-ray Detection forCapillary Electrophoresis,” J. Chromatography A, vol. 727, pp. 301-310,1996, incorporated by reference herein.

Any type of x-ray fluorescence detector can be used, such as awavelength dispersive x-ray fluorescence detector, a total reflectionx-ray fluorescence detector, or a confocal x-ray microscope, could beused.

Any target binder may be used with the present invention. Preferabletarget binders are materials that regulate biological reactions andprocesses such as glycosylation, phosphorylation, mitosis, meiosis,protein synthesis, endocytosis, cell signaling, respiration, geneexpression, cellular adhesion, membrane transport, cellular andcytoskeleton motility, DNA packaging, and the like. Such target bindersinclude, but are not limited to, enzymes, non-enzyme proteins, DNA, RNA,biological cells, and microorganisms (e.g. comprise prions, viruses,bacteria, spores) in whole or in part, and the like. Target binders mayalso include complexes of enzymes, non-enzyme proteins, DNA, RNA,biological cells, and microorganisms (e.g. comprise prions, viruses,bacteria, spores) in whole or in part, for use in competition studiesfor example. Preferably, the target binder should not flow through thesize exclusion filter but if any amount of target binder does flowthrough, the flow rate should be much less than that for potentialpharmaceutical chemical, preferably by a factor no greater than 1:100and more preferably by a factor no greater than 1:1000.

Apparatus 10 includes some means for driving fluid through the conduit,preferably a pump (not shown) such as the type of pump typically usedfor high performance liquid chromatography (HPLC). Other pumps (aperistaltic pump for example) and any other means such as voltage sourcemeans typically used for electrophoresis, vacuum source means, and othertypes of mechanical or electrical means that drive fluids throughconduit 12 can be used.

Apparatus 10 may optionally include a collector (not shown) to collectany material that passes through size exclusion filter 14, and analyzersfor analyzing these materials. An analyzer (not shown) such as a massspectrometer, gas chromatograph, liquid chromatograph, or combustionanalyzer can be used with the invention. The choice of analyzer willdepend on the nature of the chemicals and/or target binders beinganalyzed. One or more optical absorbance spectrometers (e.g.ultraviolet-visible spectrometry, infrared spectrometry), opticalfluorescence spectrometers, and other spectrometers (circular dichroismspectrometer, nuclear magnetic resonance spectrometer, surface plasmonresonance spectrometer) may be used to perform measurements onchemical-target binder complexes, on potential pharmaceutical chemicals,or on target binders present inside the conduit.

FIG. 2 shows apparatus 10 as target binder 20 flows into conduit 12 andFIG. 3 shows apparatus 10 as potential pharmaceutical chemical 26 flowsinto conduit 12. Some of the potential pharmaceutical chemical 26 flowsthrough filter 14, and some binds to the target binder 20 to formcomplex 28, which is a complex of the target binder 20 and potentialpharmaceutical chemical 26. As x-ray beam 22 is directed at filter 14,the formation of complex 28 is detected as an increase in the intensityof x-ray fluorescence 24 produced by complex 28, which is near filter14.

The invention may be used for studying the binding affinity between asmall molecule (biotin, for example) and a much larger target binder (aprotein molecule such as avidin) and the formation and release kineticsof the complex. Avidin is believed to contain from about 10 to about 16sulfur atoms per avidin molecule, and biotin has one sulfur atom permolecule. In practice, the filter from a CENTRICON™ 30 filter unit,which has a 30,000 Dalton cutoff size, is an acceptable size exclusionfilter for blocking the flow of avidin but not the flow of biotin. Thex-ray fluorescence detector can be used to quantify the signal due tosulfur as the x-ray source sends x-rays near the filter. The expectedincrease in signal intensity would indicate that more and more biotin isbinding to the avidin. After forming the complex, a solution of buffercould be flowed past the avidin-biotin complex. The expected decrease inthe sulfur signal over time could then be used to calculate the releasekinetics. The amount of bound biotin can be measured by subtracting theconcentration of biotin in the solution from the concentration of biotinmeasured using x-ray fluorescence near the filter (where theavidin/biotin complex forms). The biotin can be measured by x-rayfluorescence in a section of the conduit far enough upstream of thefilter such that the avidin concentration there is minimal. The bindingand release kinetics can be determined by comparing the amount of biotinin the complex with the rate of change of the concentration gradient ofthe biotin in solution.

The invention can also be used in pharmaceutical metabolite studies todetect dangerous metabolic byproducts of a chemical. A blood, cell,tissue, or other biological sample from an animal (a rat, for example)could provide a baseline measurement using the invention. Afteradministering the potential pharmaceutical to the animal, blood would betaken from the animal and examined for the presence of metabolites usingthe method of the invention.

The invention can also be used in DNA or RNA binding studies. A suitablesize exclusion filter that blocked the flow of DNA or RNA through theconduit of the invention would be used. The DNA or RNA could then bequantified using the x-ray fluorescence of its constituent phosphorusatoms. Another molecule of DNA, RNA, protein, or other chemical wouldthen be introduced. After quantifying the amount of this chemical, thebinding affinity could then be calculated. Binding kinetics could bedetermined as described for the biotin/avidin kinetic determinationabove

The temperature of the conduit can be controlled. This may be useful,for example, for kinetic and thermodynamic studies of binding, such asdetermining DNA melting temperatures.

In summary, the present invention provides an apparatus and method fordetecting binding events between potential pharmaceutical chemicals andtarget binders. The present invention uses x-ray fluorescence todetermine the presence and relative amounts of elements such asfluorine, chlorine, bromine, iodine, phosphorus, and sulfur, the lattertwo being important constituents of enzymes, non-enzyme proteins, DNA,and RNA. Thus, the invention provides a non-destructive method ofscreening the binding of potential pharmaceutical chemical with a targetbinder such as a protein or a nucleic acid. The invention provides theadvantage of not requiring chemicals with tags.

The foregoing description of the invention has been presented forpurposes of illustration and description and is not intended to beexhaustive or to limit the invention to the precise form disclosed, andobviously many modifications and variations are possible in light of theabove teaching.

The embodiment(s) were chosen and described in order to best explain theprinciples of the invention and its practical application to therebyenable others skilled in the art to best utilize the invention invarious embodiments and with various modifications as are suited to theparticular use contemplated. It is intended that the scope of theinvention be defined by the claims appended hereto.

1-7. (canceled)
 8. An apparatus for screening binding between a targetbinder and a chemical, comprising an x-ray translucent conduit for fluidcomprising target binder and chemical; a filter inside said conduit thatsubstantially blocks the flow of the target binder through the conduitbut not the flow of the chemical; an x-ray excitation source capable ofsending an x-ray beam through a volume of fluid inside said conduit; andan x-ray detector capable of receiving an x-ray fluorescence signalproduced from said volume of fluid inside said conduit.
 9. The apparatusof claim 8, wherein said conduit comprises an x-ray translucentcapillary tube.
 10. The apparatus of claim 8, wherein said conduitcomprises conduit walls capable of attenuating the x-ray fluorescencesignal by less than a factor of about 10,000.
 11. The apparatus of claim8, wherein the conduit comprises beryllium, boron nitride, silica, ororganic polymers.
 12. The apparatus of claim 8, further comprising apump for driving fluid through the conduit.
 13. The apparatus of claim8, further comprising a spectrometer, fluorimeter, gas chromatograph,liquid chromatograph, combustion analyzer, or sample collector situatedsubstantially downstream of said filter.